Antagonists of interleukin-15

ABSTRACT

Antagonists of mammalian interleukin-15 (&#34;IL-15&#34;) are disclosed and include muteins of IL-15 and modified IL-15 molecules that are each capable of binding to the IL-15Rα-subunit and that are incapable of transducing a signal through either the β- or γ-subunits of the IL-15 receptor complex. Also included are monoclonal antibodies against IL-15 that prevent IL-15 from effecting signal transduction through either the β- or γ-subunits of the IL-15 receptor complex. Methods of treating various disease states are disclosed, including treating allograft rejection and graft-versus-host disease.

CROSS--REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. Ser. No. 08/392,317, filed Feb.22, 1995, now U.S. Pat. No. 5,795,966.

FIELD OF THE INVENTION

The present invention relates generally to antagonists of a mammalianepithelium-derived T-cell factor polypeptide referred to herein asinterleukin-15 ("IL-15"). It more particularly relates to muteins ofIL-15, monoclonal antibodies against IL-15 and IL-15 conjugates thateach significantly reduce the ability of IL-15 to stimulate theproliferation of T-lymphocytes in an in vitro CTLL assay. Also includedin the invention are methods for treating various disease states inmammals where a reduction in IL-15 activity is desired.

BACKGROUND OF THE INVENTION

Interleukin-15 is a known T-cell growth factor that can supportproliferation of an IL-2-dependent cell line, CTLL-2. IL-15 was firstreported by Grabstein et al., in Science, 264:965 (1994) as a secretedcytokine comprising a 162-amino acid precursor polypeptide that containsa 48-amino acid leader sequence that results in a 114-amino acid matureprotein. Grabstein et al. also describe the cloning of the full-lengthhuman cDNA encoding the 162-amino acid precursor, which contains a 316bp 5' noncoding region and a 486 bp open reading frame (or a 489 bp openreading frame when including the 3 bp for the stop codon) and a 400 bp3' noncoding region.

IL-15 shares many properties with IL-2. These properties includeproliferation and activation of human and murine T cells, the inductionof lymphokine activated killer cell (LAK) activity, natural killer cell(NK) activity, and cytotoxic T lymphocytes (CTL) activity, andcostimulation of B cell proliferation and differentiation.

Additionally, IL-15 and IL-2 are structurally homologous molecules thatare able to bind to at least three distinct receptor subunits on the Tcell membrane surface. IL-2 receptors contain at least three subunits,α, β and γ (Toshikazu et al., Science, 257:379 (1992)). Both IL-15 andIL-2 share binding to a common β--γ subunit complex, while each of IL-15and IL-2 bind to a specific α-receptor subunit (IL-15Rα and IL-2Rα,respectively). Recently, the IL-15Rα was discovered and is the subjectof copending application Ser. No. 08/300,903, now U.S. Pat. No.5,591,630. Antibodies directed against the α-chain of the IL-2 receptor(anti-IL-2Rα) have no effect on IL-15 binding (Grabstein et al., Id.).Antibodies directed against the β-subunit of the IL-2 receptor, i.e.,TU27, TU11, or Mikβ1, however, are able to block the activity of IL-15,suggesting that IL-15 uses the β-subunit for signaling. Similarly, theγ-chain of the IL-2 receptor is required for signal transduction (Giriet al., EMBO J., 13:2822 (1994)). The combination of the β and theγ-subunits of the IL-15 receptor complex, but neither subunit alone,bound IL-15 on transfected COS cells.

Certain disease states and physiological conditions are mediated by Tcells. Such diseases include organ transplant rejection, graft versushost disease, autoimmune disease, rheumatoid arthritis, inflammatorybowel disease, dermatologic disorders, insulin-dependent diabetesmellitus, ocular disorders and idiopathic nephrotic syndrome/idiopathicmembranous nephropathy. Indeed, allograft rejection andgraft-versus-host disease (GVHD) have been associated with increasedIL-2 receptor expression. T cells activated in response to foreignhistocompatibility antigens appear to express the IL-2 receptor complex.Various therapies have been proposed and studied. For example, Tinubu etal. (J. Immunol., 153:4330 (1994)), reported that the anti-IL-2Rβmonoclonal antibody, Mikβ1, prolongs primate cardiac allograft survival.There is an increase in IL-2Rβ-subunit expression on CD4-andCD8-expressing cells in association with acute allograft rejection,which indicates that the IL-2Rβ-subunit expression seems to increase onalloreactive T cells. See, for example, Niguma et al., Transplantation,52:296 (1991).

However, prior to the present invention, there have been no therapiesthat focused on the IL-15 ligand-receptor interaction as a means oftreating GVHD or in promoting allograft survival.

SUMMARY OF THE INVENTION

The invention is directed to IL-15 antagonists and a method of using theantagonists for treatment of human disease. In particular, suchtreatment includes promoting allograft survival in mammals and treatingGVHD. The IL-15 antagonists are effective by preventing IL-15 fromtransducing a signal to a cell through either the β- or γ-subunits ofthe IL-15 receptor complex. thereby antagonizing IL-15's biologicalactivity. Certain of the antagonists according to the invention mayinterfere with the binding of IL-15 to the β- or γ-subunits of the IL-15receptor complex, while not substantially interfering with the bindingof IL-15 to IL-15Rα.

Antagonists according to the invention include muteins of mature, ornative, IL-15, wherein IL-15 has been mutagenized at one or more aminoacid residues or regions that play a role in binding to the β- orγ-subunit of the IL-15 receptor complex. Such muteins prevent IL-15 fromtransducing a signal to the cells through either of the β- or γ-subunitsof the IL-15 receptor complex, while maintaining the high affinity ofIL-15 for the IL-15Rα. Typically, such muteins are created by additions,deletions or substitutions at key positions, for example, Asp⁵⁶ orGln¹⁵⁶ of simian and human IL-15 as shown in SEQ ID NOS: 1 and 2,respectively. It is believed that the Asp⁵⁶ affects binding with theβ-subunit and that the Gln¹⁵⁶ affects binding with the γ-subunit of theIL-15 receptor complex.

In addition, the invention encompasses monoclonal antibodies thatimmunoreact with mature IL-15 and prevent signal transduction to theIL-15 receptor complex.

Further included in the scope of the invention are modified IL-15molecules that retain the ability to bind to the IL-15Rα, but havesubstantially diminished or no affinity for the β- and/or γ-subunits ofthe IL-15 receptor complex. Modified IL-15 molecules can take any formas long as the modifications are made in such a manner as to interferewith or prevent binding, usually by modification at or near the targetbinding site. Examples of such modified IL-15 molecules include matureIL-15 or a mutein of IL-15 that is covalently conjugated to one or morechemical groups that sterically interfere with the IL-15/IL- 15 receptorbinding. For example, mature IL-15 may contain site-specificglycosylation or may be covalently bound to groups such as polyethyleneglycol (PEG), monomethoxyPEG (mPEG), dextran, polyvinylpyrrolidone(PVP), polyvinyl alcohol (PVA), poly amino acids such as poly-L-lysineor polyhistidine, albumin, gelatin at specific sites on the IL-15molecule that can interfere with binding of IL-15 to the β- or γ-chainsof the IL-15 receptor complex, while maintaining the high affinity ofIL-15 for the IL-15Rα. By taking advantage of the steric hindranceproperties of the group, binding to specific receptor subunits can beantagonized. Other advantages of conjugating chains of PEG to proteinssuch as IL-2, GM-CSF, asparaginase, immunoglobulins, hemoglobin, andothers are known in the art. For example, it is known that PEG prolongscirculation half-lives in vivo (see, Delgado, et al., Crit. Rev. Ther.Drug Carr. Syst., 9:249 (1992)), enhances solubility (see, Katre, etal., Proc. Natl. Acad. Sci., 84:1487 (1987)) and reduces immunogenicity(see. Katre, N. V., Immunol. 144:209 (1990)).

The invention also is directed to the use of the antagonists in a methodof treating a disease or condition in which a reduction in IL-15activity on T cells is desired. Such diseases include organ transplantrejection, graft versus host disease, autoimmune disease, rheumatoidarthritis, inflammatory bowel disease, dermatologic disorders,insulin-dependent diabetes mellitus, ocular disorders and idiopathicnephrotic syndrome/idiopathic membranous nephropathy. In particular, inallograft rejection, IL-15 activity may lead to a host immune responseagainst the graft and eventually rejection. Similarly, in GVHD, thegraft, typically a bone marrow transplant, imparts an immune responseagainst the host. Suppression of such activities by the IL-15antagonists according to the invention may be advantageous in promotingand prolonging graft survival, and in treating GVHD.

Various investigators have reported the prolongation of graft survivalby using antibodies, such as anti-TAC, an anti-human IL-2 α-receptormonoclonal antibody. See Reed et al., Transplantation, 47:55-59 (1989),wherein anti-TAC is shown to have improved primate renal allografttransplantation. Also, Brown et al., Proc. Natl. Acad. Sci., 88:2663(1991) describe the use of humanized anti-TAC in prolonging primatecardiac allograft survival. Kirkman et al., Transplantation, 51:107(1991), also describe a clinical trial involving anti-TAC in preventingearly allograft rejection. Since IL-15 possesses many biologicalactivities similar to IL-2, and indeed, shares certain receptor subunitswith IL-2, interfering with a deleterious activity of IL-15 in diseasedconditions has distinct therapeutic potential.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to an antagonist of IL-15 activity thatinterferes with the signal transduction of IL- 15 through its receptorcomplex. In particular, the IL-15 antagonists of the invention areselected from the group consisting of (a) a mutein of mature, or native,IL-15 capable of binding to the α-subunit of the IL-15 receptor andincapable of transducing a signal through the β- and/or γ-subunits ofthe IL-15 receptor complex; (b) a monoclonal antibody against IL-15 thatprevents IL-15 from effecting signal transduction through the β- and/or-γsubnits of the IL-15 receptor complex: and (c) an IL-15 molecule thatis covalently bonded with a chemical group that interferes with theability of IL-15 to effect a signal transduction through either the β-or γ-subunits of the IL-15 receptor complex, but does not interfere withIL-15 binding to IL-15Rα. Also included in the scope of the presentinvention are the DNAs that encode the muteins described above.

As used herein, "Recombinant DNA technology" or "recombinant" refers totechniques and processes for producing specific polypeptides frommicrobial (e.g., bacterial, insect or yeast) or mammalian cells ororganisms (e.g., transgenics) that have been transformed or transfectedwith cloned or synthetic DNA sequences to enable biosynthesis ofheterologous peptides. Native glycosylation patterns will only beachieved with mammalian cell expression systems. Yeast provide adistinctive glycosylation pattern. Prokaryotic cell expression (e.g., E.Coli) will generally produce polypeptides without glycosylation.

A "nucleotide sequence" refers to a polynucleotide in the form of aseparate fragment or as a component of a larger DNA construct, that hasbeen derived from DNA or RNA isolated at least once in substantiallypure form (i.e., free of contaminating endogenous materials) and in aquantity or concentration enabling identification, manipulation, andrecovery of its component nucleotide sequences by standard biochemicalmethods (such as those outlined in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989)). Such sequences are preferably provided in the formof an open reading frame uninterrupted by internal nontranslatedsequences, or introns, that are typically present in eukaryotic genes.Sequences of non-translated DNA may be present 5' or 3' from an openreading frame, where the same do not interfere with manipulation orexpression of the coding regions.

"Recombinant expression vector" refers to a plasmid comprising atranscriptional unit comprising an assembly of (1) a genetic element orelements having a regulatory role in gene expression, for example,promoters or enhancers, (2) a structural or coding sequence that encodesIL-15 or an IL-15 mutein, and (3) appropriate transcription andtranslation initiation sequences and, if desired, termination sequences.The representative examples of various regulatory elements that can beused are discussed below (see Recombinant DNA Techniques). Structuralelements intended for use in yeast expression systems preferably includea leader sequence enabling extracellular secretion of translatedpolypeptide by a yeast host cell. Alternatively, in a bacterialexpression system, the recombinant polypeptide may include a N-terminalmethionine residue. The N-terminal methionine residue may besubsequently cleaved from the expressed recombinant polypeptide toprovide a product suitable for further purification.

"Recombinant microbial expression system" refers to a substantiallyhomogeneous monoculture of suitable host microorganisms, for example,bacteria, such as E. coli, or yeast, such as S. cerevisiae, that havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit as a component of aresident plasmid. Generally, host cells constituting a recombinantmicrobial expression system are the progeny of a single ancestraltransformed cell. Recombinant microbial expression systems will expressheterologous polypeptides upon induction of the regulatory elementslinked to a structural nucleotide sequence to be expressed.

"IL-15 mutein" or "muteins of IL-15" refer to the mature, or native,simian IL-15 molecules having the sequence of amino acids 49-162 of SEQID NO: 1 or human IL-15 molecules having the sequence of amino acids49-162 of SEQ ID NO: 2, that have been mutated in accordance with theinvention in order to produce an antagonist of IL-15. Such IL-15 muteinsare capable of binding to the IL-15Rα subunit, and are incapable oftransducing a signal through the β- or γ-subunits of the IL-15 receptorcomplex.

Preparation of IL-15

Human or simian IL-15 can be obtained according to the proceduresdescribed by Grabstein et al., Science, 264:965 (1994), which has beenincorporated herein by reference, or by conventional procedures such aspolymerase chain reaction (PCR). A deposit of human IL-15 cDNA was madewith the American Type Culture Collection, Rockville, Md. USA (ATCC) onFeb. 19, 1993 and assigned accession number 69245. The deposit was named"I41-hETF." The deposit was made according to the terms of the BudapestTreaty.

IL-15 Muteins

There are many possible mutations of IL-15 that can produce antagonists.Such mutations can be made at specific amino acid sites believed to beresponsible for β- or γ-subunit necessary for β- or γ-subunit signaling.Typically, mutations may be made as additions, substitutions ordeletions of amino acid residues. Preferably, substitution and deletionmuteins are preferred with substitution muteins being most preferred.

It is believed that the Asp⁵⁶ affects binding with the β-subunit andthat the Gln¹⁵⁶ affects binding with γ-subunit of the IL-15 receptorcomplex. Adding or substituting other naturally-occurring amino acidresidues near or at sites Asp⁵⁶ and Gln¹⁵⁶ can affect the binding ofIL-15 to either or both of the β- or γ-subunits of the IL-15 receptorcomplex. Indeed, removing the negatively-charged aspartic acid residueand replacing it with another negatively-charged residue may not be aseffective at blocking receptor binding as if the aspartic acid werereplaced with a positively-charged amino acid such as arginine, oruncharged residues such as serine or cysteine.

Recombinant production of an IL-15 mutein first requires isolation of aDNA clone (i.e., cDNA) that encodes an IL-15 mutein. cDNA clones arederived from primary cells or cell lines that express mammalian IL-15polypeptides. First total cell mRNA is isolated then a cDNA library ismade from the mRNA by reverse transcription. A cDNA clone may beisolated and identified using the DNA sequence information providedherein to design a cross-species hybridization probe or PCR primer asdescribed above. Such cDNA clones have the sequence of nucleic acids1-486 of SEQ ID NO: 1 and SEQ ID NO: 2.

The isolated cDNA is preferably in the form of an open reading frameuninterrupted by internal nontranslated sequences, or introns. GenomicDNA containing the relevant nucleotide sequences that encode mammalianIL-15 polypeptides can also be used as a source of genetic informationuseful in constructing coding sequences. The isolated cDNA can bemutated utilizing techniques known in the art to provide IL-15antagonist activity. Below, example 1 describes a specific method thatcan be used to prepare the IL-15 muteins.

Equivalent DNA constructs that encode various additions or substitutionsof amino acid residues or sequences, or deletions of terminal orinternal residues or sequences not needed for activity are encompassedby the invention. For example, N-glycosylation sites in IL-15 can bemodified to preclude glycosylation, allowing expression of a reducedcarbohydrate analog in mammalian and yeast expression systems.N-glycosylation sites in eukaryotic polypeptides are characterized by anamino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Yis Ser or Thr. The simian IL-15 protein comprises two such triplets, atamino acids 127-129 and 160-162 of SEQ ID NO: 1. The human IL-15 proteincomprises three such triplets, at amino acids 119-121, 127-129 and160-162 of SEQ ID NO: 2. Appropriate substitutions, additions ordeletions to the nucleotide sequence encoding these triplets will resultin prevention of attachment of carbohydrate residues at the Asn sidechain. Alteration of a single nucleotide, chosen so that Asn is replacedby a different amino acid, for example, is sufficient to inactivate anN-glycosylation site. Known procedures for inactivating N-glycosylationsites in proteins include those described in U.S. Pat. No. 5,071,972 andEP 276,846. hereby incorporated by reference.

Recombinant expression vectors include synthetic or cDNA-derived DNAfragments encoding an IL-15 mutein. The DNA encodina an IL-15 mutein isoperably linked to a suitable transcriptional or translationalregulatory or structural nucleotide sequence, such as one derived frommammalian, microbial, viral or insect genes. Examples of regulatorysequences include, for example, a genetic sequence having a regulatoryrole in gene expression (e.g., transcriptional promoters or enhancers),an optional operator sequence to control transcription. a sequenceencoding suitable mRNA ribosomal binding sites, and appropriatesequences that control transcription and translation initiation andtermination. Nucleotide sequences are operably linked when theregulatory sequence functionally relates to the structural gene. Forexample, a DNA sequence for a signal peptide (secretory leader) may beoperably linked to a structural gene DNA sequence for an IL-15 mutein ifthe signal peptide is expressed as part of a precursor amino acidsequence and participates in the secretion of an IL-15 mutein. Further,a promoter nucleotide sequence is operably linked to a coding sequence(e.g., structural gene DNA) if the promoter nucleotide sequence controlsthe transcription of the structural gene nucleotide sequence. Stillfurther, a ribosome binding site may be operably linked to a structuralgene nucleotide coding sequence (e.g. IL-15 mutein) if the ribosomebinding site is positioned within the vector to encourage translation.

Suitable host cells for expression of an IL-15 mutein includeprokaryotes, yeast or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Suitable prokaryotichosts cells for transformation include, for example, E. coli, Bacillussubtilis, Salmonella typhimurium, and various other species within thegenera Pseudomonas, Streptomyces, and Staphylococcus. As discussed ingreater detail below, examples of suitable host cells also include yeastsuch as S. cerevisiae, a mammalian cell line such as Chinese HamsterOvary (CHO) cells, or insect cells. Cell-free translation systems couldalso be employed to produce an IL-15 mutein using RNAs derived from theDNA constructs disclosed herein. Appropriate cloning and expressionvectors for use with bacterial, insect, yeast, and mammalian cellularhosts are described, for example, in Pouwels et al. Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985.

When an IL-15 mutein is expressed in a yeast host cell, the nucleotidesequence (e.g., structural gene) that encodes an IL-15 mutein mayinclude a leader sequence. The leader sequence may enable improvedextracellular secretion of translated polypeptide by a yeast host cell.

IL-15 muteins may be expressed in yeast host cells, preferably from theSaccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, suchas Pichia or Kluyveromyces, may also be employed. Yeast vectors willoften contain an origin of replication sequence from a 2μ yeast plasmid,an autonomously replicating sequence (ARS), a promoter region, sequencesfor polyadenylation, and sequences for transcription termination.Preferably, yeast vectors include an origin of replication sequence andselectable marker. Suitable promoter sequences for yeast vectors includepromoters for metallothionein. 3-phosphoglycerate kinase (Hitzeman etal., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess etal., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochlem.17:4900, 1978), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoalucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EP-A-73,657.

Yeast vectors can be assembled, for example, using DNA sequences frompBR322 for selection and replication in E. coli (Amp^(r) gene and originof replication). Other yeast DNA sequences that can be included in ayeast expression construct include a glucose-repressible ADH2 promoterand β-factor secretion leader. The ADH2 promoter has been described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). The east α-factor leader sequence directs secretion ofheterologous polypeptides. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330, 1984. A leader sequence may be modifiednear its 3' end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those skilled in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a "rich" medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/mluracil. Repression of the ADH2 promoter is lost when glucose isexhausted from the medium.

Alternatively, in a prokaryotic host cell, such as E. coli, the IL-15mutein may include an N-terminal methionine residue to facilitateexpression of the recombinant polypeptide in a prokaryotic host cell.The N-terminal Met may be cleaved from the expressed recombinant IL-15mutein.

The recombinant expression vectors carrying the recombinant IL-15 muteinstructural gene nucleotide sequence are transfected or transformed intoa suitable host microorganism or mammalian cell line.

Expression vectors transfected into prokaryotic host cells generallycomprise one or more phenotypic selectable markers. A phenotypicselectable marker is, for example, a gene encoding proteins that conferantibiotic resistance or that supply an autotrophic requirement, and anorigin of replication recognized by the host to ensure amplificationwithin the host. Other useful expression vectors for prokarvotic hostcells include a selectable marker of bacterial origin derived fromcommercially available plasmids. This selectable marker can comprisegenetic elements of the cloning vector pBR322 (ATCC 37017). pBR322contains genes for ampicillin and tetracycline resistance and thusprovides simple means for identifying transformed cells. The pBR322"backbone" sections are combined with an appropriate promoter and aIL-15 mutein structural gene sequence. Other commercially availablevectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).

Promoter sequences are commonly used for recombinant prokaryotic hostcell expression vectors. Common promoter sequences include β-lactamase(penicillinase), lactose promoter system (Chang et al., Nature 275:615,1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp)promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EPA36,776) and tac promoter (Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, (1989)). Aparticularly useful prokaryotic host cell expression system employs aphage λP_(L) promoter and a cI857ts thermolabile repressor sequence.Plasmid vectors available from the American Type Culture Collection thatincorporate derivatives of the λP_(L) promoter include plasmid pHUB2(resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident inE. coli RR1 (ATCC 53082)).

Mammalian or insect host cell culture systems also could be employed toexpress recombinant IL-15 muteins. Examples of suitable mammalian hostcell lines include the COS-7 lines of monkey kidney cells (Gluzman etal., Cell 23:175, (1981); ATCC CRL 1651), L cells, C127 cells, 3T3 cells(ATCC CCL 163), CHO cells, HeLa cells (ATCC CCL 2), and BHK (ATCC CRL10) cell lines. Suitable mammalian expression vectors includenontranscribed elements such as an origin of replication, a promotersequence, an enhancer linked to the structural gene, other 5' or 3'flanking nontranscribed sequences, such as ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

Transcriptional and translational control sequences in mammalian hostcell expression vectors may be provided by viral sources. For example,commonly used mammalian cell promoter sequences and enhancer sequencesare derived from Polyoma, Adenovirus 2. Simian Virus 40 (SV40), andhuman cytomegaloyirus. DNA sequences derived from the SV40 viral genome,for example, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a structural gene sequence in a mammalianhost cell. Viral early and late promoters are particularly usefulbecause both are easily obtained from a viral genome as a fragment thatmay also contain a viral origin of replication (Fiers et al., Nature273:113, 1978). Smaller or larger SV40 fragments may also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Exemplary mammalian expression vectors can be constructed as disclosedby Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). Additional usefulmammalian expression vectors are described in U.S. patent applicationSer. No. 07/480,694 filed Feb. 14, 1990 and U.S. Pat. No. 5,350,683.

Purification of Recombinant IL-15 Muteins

In general, IL-15 mutein polypeptides may be prepared by culturingtransformed host cells under culture conditions necessary to expressIL-15 mutein polypeptides. The resulting expressed mutein may then bepurified from culture media or cell extracts. An IL-15 mutein may beconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. With or without the concentration step, the culture media can beapplied to a purification matrix such as a hydrophobic chromatographymedium. Phenyl Sepharose® CL-4B (Pharmacia) is the preferred medium.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, gel filtrationmedium can be used.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant butyl or other aliphatic groups, can beemployed to further purify IL-15 muteins. An S Sepharose (Pharmacia)cation exchange column may also be employed as a final buffer exchangestep. Some or all of the foregoing conventional purification steps, invarious combinations, can also be employed to provide a substantiallyhomogeneous recombinant protein.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant if asoluble polypeptide, followed by one or more concentration, salting-out,ion exchange or size exclusion chromatography steps. Finally, RP-HPLCcan be employed for final purification steps. Microbial cells can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents.

Transformed yeast host cells are preferably employed to express an IL-15mutein as a secreted polypeptide. Secreted recombinant polypeptide froma yeast host cell fermentation can be purified by methods analogous tothose disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal etal. describe two sequential, reversed-phase HPLC steps for purificationof recombinant human IL-2 on a preparative HPLC column.

Preferably, a mutein of IL-15 is used wherein at least one of the aminoacid residues Asp⁵⁶ or Gln¹⁵⁶ of IL-15 (simian IL-15 having the sequenceof amino acid residues 49-162 shown in SEQ ID NO: 1 or human IL-15having the sequence of amino acid residues 49-162 shown in SEQ ID NO: 2)is deleted or substituted with a different naturally-occurring aminoacid residue. Any combination of substitutions and/or deletions can bemade. For example, Asp⁵⁶ can be deleted while Gln¹⁵⁶ is substituted withany other amino acid, or both Asp⁵⁶ and Gln¹⁵⁶ are each substituted withthe same or different amino acid moiety. Further, Asp⁵⁶ can besubstituted with any amino acid while Gln¹⁵⁶ is deleted. Generally,substitution muteins are preferred, and more preferred are those that donot severely affect the natural folding of the IL-15 molecule.Substitution muteins preferably include those wherein Asp⁵⁶ issubstituted by serine or cysteine; or wherein Gln¹⁵⁶ is substituted byserine or cysteine, or wherein both Asp⁵⁶ and Gln¹⁵⁶ are eachsubstituted with a serine or cysteine. Examples of deletion muteinsinclude those wherein Asp⁵⁶ and Gln¹⁵⁶ of mature IL-15 are both deleted;wherein only Asp⁵⁶ is deleted; or wherein only Gln¹⁵⁶ is deleted. It ispossible that other amino acid residues in the region of either Asp⁵⁶and Gln¹⁵⁶ can be substituted or deleted and still have an effect ofpreventing signal transduction through either or both of the β- orγ-subunits of the IL-15 receptor complex. Therefore, the inventionfurther encompasses muteins wherein amino acid residues within theregion of Asp⁵⁶ and Gln¹⁵⁶ are either substituted or deleted, and thatpossess IL-15 antagonist activity. Such muteins can be made using themethods described herein and can be assayed for IL-15 antagonistactivity using conventional methods. Further description of a methodthat can be used to create the IL-15 muteins according to the inventionis provided in Example 1.

Conjugated IL-15 Molecules and IL-15 Muteins

The mature IL-15 polypeptides disclosed herein (mature simian IL-15comprising the sequence of amino acids 49-162 of SEQ ID NO: 1 and maturehuman IL-15 having the sequence of amino acid residues 49-162 shown inSEQ ID NO: 2), as well as the IL-15 muteins, may be modified by formingcovalent or aggregative conjugates with other chemical moieties. Suchmoieties can include PEG, mPEG, dextran, PVP, PVA, polyamino acids suchas poly-L-lysine or polyhistidine, albumin and gelatin at specific siteson the IL-15 molecule that can interfere with binding of IL-15 to the β-or γ-chains of the IL-15 receptor complex, while maintaining the highaffinity of IL-15 for the IL-15Rα. Additionally, IL-15 can bespecifically glycosylated at sites that can interfere with binding ofIL-15 to the β- or γ-chains of the IL-15 receptor complex, whilemaintaining the high affinity of IL-15 for the IL-15Rα. Preferred groupsfor conjugation are PEG, dextran and PVP. Most preferred for use in theinvention is PEG, wherein the molecular weight of the PEG is preferablybetween about 1,000 to about 20,000. A molecular weight of about 5000 ispreferred for use in conjugating IL-15, although PEG molecules of otherweights would be suitable as well. A variety of forms of PEG aresuitable for use in the invention. For example, PEG can be used in theform of succinimidyl succinate PEG (SS-PEG) which provides an esterlinkage that is susceptible to hydrolytic cleavage in vivo, succinimidylcarbonate PEG (SC-PEG) which provides a urethane linkage and is stableagainst hydrolytic cleavage in vivo, succinimidyl propionate PEG(SPA-PEG) provides an ether bond that is stable in vivo, vinyl sulfonePEG (VS-PEG) and maleimide PEG (Mal-PEG) all of which are commerciallyavailable from Shearwater Polymers, Inc. (Huntsville, AL). In general,SS-PEG, SC-PEG and SPA-PEG react specifically with lysine residues inthe polypeptide, whereas VS-PEG and Mal-PEG each react with freecysteine residues. However, Mal-PEG is prone to react with lysineresidues at alkaline pH. Preferably, SC-PEG and VS-PEG are preferred,and SC-PEG is most preferred due to its in vivo stability andspecificity for lysine residues.

The PEG moieties can be bonded to IL-15 in strategic sites to takeadvantage of PEG's large molecular size. As described above, PEGmoieties can be bonded to IL-15 by utilizing lysine or cysteine residuesnaturally occurring in the protein or by site-specific PEGylation. Onemethod of site specific PEGylation is through methods of proteinengineering wherein cysteine or lysine residues are introduced intoIL-15 at specific amino acid locations. The large molecular size of thePEG chain(s) conjugated to IL-15 is believed to block the region ofIL-15 that binds to the β- and/or γ-subunits but not the α-subunit ofthe IL-15 receptor complex. Conjugations can be made by a simpleaddition reaction wherein PEG is added to a basic solution containingIL-15. Typically, PEGylation is carried out at either (1) about pH 9.0and at molar ratios of SC-PEG to lysine residue of approximately 1:1 to100:1, or greater, or (2) at about pH 7.0 and at molar ratios of VS-PEGto cysteine residue of approximately 1:1 to 100:1, or greater.

Characterization of the conjugated PEGylated IL-15 molecules can beperformed by SDS-PAGE on a 4-20% gradient polyacrylamide gel, availablefrom Novex Corp., San Diego, Calif. Conventional silver staining may beemployed, or conventional Western blotting techniques can be utilizedfor highly PEGylated proteins that are not visualized easily by silverstaining. Purification of the PEGylated IL-15 molecules can be performedusing size exclusion chromatography, dialysis, ultrafiltration oraffinity purification.

The extent of modification and heterogeneity of PEGylated IL-15 can bedetermined using conventional matrix assisted laser desorptionionization mass spectrometry (MALDI). Since human IL-15 has a molecularweight of about 13,000 and by using PEG having a molecular weight of5000, MALDI indicates that preparations weighing 13,000 are unPEGylated,those weighing 18,000 indicate that 1 molecule of IL-15 is bonded to onePEG molecule; those weighing 23,000 signify that one IL-15 molecule isbound with two PEG molecules, etc.

Monoclonal Antibodies Against IL-15

Alternatively, an antagonist according to the invention can take theform of a monoclonal antibody against IL-15 that interferes with thebinding of IL-15 to any of the α-, β- or γ-subunits of the IL-15receptor complex. Within one aspect of the invention. IL-15, includingderivatives thereof, as well as portions or fragments of these proteinssuch as IL-15 peptides, can be used to prepare antibodies thatspecifically bind to IL-15. Within the context of the invention, theterm "antibodies" should be understood to include polyclonal antibodies,monoclonal antibodies, fragments thereof such as F(ab')2 and Fabfragments, as well as recombinantly produced binding partners. Theaffinity of a monoclonal antibody or binding partner may be readilydetermined by one of ordinary skill in the art (see Scatchard, Ann. N.Y.Acad. Sci., 51: 660-672 (1949)). Specific examples of such monoclonalantibodies are provided in Example 2 herein.

In general, monoclonal antibodies against IL-15 can be generated usingthe following procedure. Purified IL-15, a fragment thereof, syntheticpeptides or cells that express IL-15 can be used to generate monoclonalantibodies against IL-15 using techniques known per se, for example,those techniques described in U.S. Pat. No. 4,411,993. Briefly, mice areimmunized with IL-15 as an immunogen emulsified in complete Freund'sadjuvant or RIBI adjuvant (RIBI Corp., Hamilton, Mont.), and injected inamounts ranging from 10-100 μg subcutaneously or intraperitoneally. Tento twelve days later, the immunized animals are boosted with additionalIL-15 emulsified in incomplete Freund's adjuvant. Mice are periodicallyboosted thereafter on a weekly to bi-weekly immunization schedule. Serumsamples are periodically taken by retro-orbital bleeding or tail-tipexcision to test for IL-15 antibodies by dot blot assay, ELISA(Enzyme-Linked Immunosorbent Assay) or inhibition of IL-15 activity onCTLL-2 cells.

Following detection of an appropriate antibody titer, positive animalsare provided an additional intravenous injection of IL-15 in saline.Three to four days later, the animals are sacrificed, spleen cellsharvested, and spleen cells are fused to a murine myeloma cell line,e.g., NS1 or preferably P3×63Ag8.653 (ATCC CRL 1580). Fusions generatehybridoma cells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused myeloma cells and myeloma hybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified IL-15 by adaptations of the techniques disclosed in Engvall etal., Immunochem. 8:871, 1971 and in U.S. Pat. No. 4,703,004. A preferredscreening technique is the antibody capture technique described inBeckmann et al., (J. Immunol. 144:4212, 1990). Positive hybridoma cellscan be injected intraperitoneally into syngeneic Balb/c mice to produceascites containing high concentrations of anti-IL-15 monoclonalantibodies. Alternatively, hybridoma cells can be grown in vitro inflasks or roller bottles by various techniques. Monoclonal antibodiesproduced in mouse ascites can be purified by ammnonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to protein A orprotein G can also be used, as can affinity chromatography based uponbinding to IL-15.

Other "antibodies" can be prepared utilizing the disclosure providedherein, and thus fall within the scope of the invention. Procedures usedto generate humanized antibodies can be found in U.S. Pat. No. 4,816,567and WO 94/10332; procedures to generate microbodies can be found in WO94/09817; and procedures to generate transgenic antibodies can be foundin GB 2 272 440, all of which are incorporated herein by reference.

To determine which monoclonal antibodies are antagonists, use of ascreening assay is preferred. A CTLL-2 proliferation assay is preferredfor this purpose. See, Gillis and Smith, Nature 268:154 (1977), which isincorporated herein by reference.

The antagonists according to the invention find use, as described aboveand in more detail below, in promoting allograft survival and intreating patients with graft versus host disease. Another credible usefor the antagonists include the treatment of late phase HTLV (humanT-cell lymphotrophic virus) I-induced adult T-cell leukemia-lymphoma.See Burton et al., Proc. Natl. Acad. Sci., 91:4935 (1994). Othercredible uses include ability to prevent B cell or T-cell stimulation invitro, study receptor-ligand interaction, in diagnostic kits forinfectious disease and disorders of the gastrointestinal tract. Byvirtue of the activity of the antagonists according to the invention,new methods of treating certain diseases are within the scope of theinvention. For example, there is disclosed a method for preventingallograft rejection in a patient in need thereof, and a method oftreating GVHD in a patient in need thereof, each method comprising thestep of administering a pharmaceutical composition comprising an amountof an IL-15 antagonist effective to inhibit IL-15 activity, and apharmaceutically acceptable carrier or diluent. Similar methods areuseful for treating other diseases whereby the target cells (the cellsthat are believed to be primarily responsible for the diseasedcondition, or a symptom of the diseased condition) are expressing theIL-15) receptor complex and where a blockade or inhibition of signaltransduction through the β- or γ-subunits of the IL-15 receptor isdesired. Such disease states may be treatable with the antagonists ofthe invention upon learning that the target cells express the IL-15receptor complex. Indeed, in addition to GVHD and allograft rejection,such disease states may include, for example, lymphomas, carcinomas,leukemias, rhabdosarcomas, and certain autoimmune disorders such asrheumatoid arthritis. The fact that the foregoing list is not exhaustiveof all disease states wherein the target cells express the requiredIL-15-receptor complex, should not be construed as limiting the scope ofthe invention.

As described above, another embodiment of the invention is the nucleicacids that encode the IL-15 muteins of the invention. Such nucleic acidscomprise either RNA or the cDNA having the nucleotide sequence from 144to 486 of SEQ ID NO: 1 and 144 to 486 of SEQ ID NO: 2. Further withinthe scope of the invention are expression vectors that comprise a cDNAencoding an IL-15 mutein and host cells transformed or transfected withsuch expression vector. Transformed host cells are cells that have beentransformed or transfected with a recombinant expression vector usingstandard procedures. Expressed mammalian IL-15 will be located withinthe host cell and/or secreted into culture supernatant, depending uponthe nature of the host cell and the gene construct inserted into thehost cell. Pharmaceutical compositions comprising any of theabove-described IL-15 antagonists also are encompassed by thisinvention.

Administration of Antagonists of IL-15

The present invention provides methods of using pharmaceuticalcompositions comprising an effective amount of IL-15 antagonist in asuitable diluent or carrier. An IL-15 antagonist of the invention can beformulated according to known methods used to prepare pharmaceuticallyuseful compositions. An IL-15 antagonist can be combined in admixture.either as the sole active material or with other known active materials,with pharmaceutically suitable diluents (e.g. Tris-HCl, acetate,phosphate), preservatives (e.g., Thimerosal, benzyl alcohol, parabens),emulsifiers, solubilizers, adjuvants and/or carriers. Suitable carriersand their formulations are described in Remington's PharmaceuticalSciences, 16th ed. 1980, Mack Publishing Co. In addition, suchcompositions can contain an IL-15 antagonist complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporatedinto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts or spheroblasts. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance of an IL-15 antagonist. An IL-15antagonist can also be conjugated to antibodies against tissue-specificreceptors, ligands or antigens, or coupled to ligands of tissue-specificreceptors.

The IL-15 antagonist of the invention can be administered topically,parenterally, rectally or by inhalation. The term "parenteral" includessubcutaneous injections, intravenous, intramuscular, intracisternalinjection, or infusion techniques. These compositions will typicallycontain an effective amount of an IL-15 antagonist, alone or incombination with an effective amount of any other active material. Suchdosages and desired drug concentrations contained in the compositionsmay vary depending upon many factors, including the intended use,patient's body weight and age, and route of administration. Preliminarydoses can be determined according to animal tests, and the scaling ofdosages for human administration can be performed according toart-accepted practices.

In addition to the above, the following. examples are provided toillustrate particular embodiments and not to limit the scope of theinvention.

EXAMPLE 1 Muteins of IL-15

This example describes a method for obtaining muteins of mature, ornative, IL-15 that function as antagonists of IL-15. IL-15, like IL-2,is able to bind to and signal through the IL-2Rβγ complex, and as such,is proposed to share structural similarities to IL-2. The equivalentresidues in IL-15 that have previously been shown in IL-2 to be criticalfor interaction with the IL-2Rβ- and γ-chain (Zurawski, et al., EMBO J.,12(13):5113 (1993)) were determined by best-fit sequence alignment to beaspartic acid, residue 56 (Asp) for the β-chain, and glutamine, residue156 (Gln) for the γ-chain (amino acid numbering is based on the sequenceof the peptide as shown by amino acid residues 1-162 of SEQ ID NOS: 1and 2).

Oligonucleotide primers were designed that would amplify human IL-15 andintroduce a codon encoding either a serine or a cysteine at eitherresidue 56 or 156. Two separate rounds of PCR amplification wereperformed for the construction of each mutant (see diagram below). Inthe primary PCR reaction, amplification was with primer pairs thateither introduced the appropriate mutation, or amplified the maturesequence. In the secondary PCR reaction, material from the first roundwas reamplified with a primer set that introduced restriction sites forcloning into the pαADH2 yeast expression vector pIXY456. See, Price etal., Gene, 55:287 (1987) and Price et al., Meth. Enzym. 185:308 (1990).##STR1## The table below lists the pairs of oligonucleonde primers usedfor the primary amplification of each mutein. The oligonucleotidesNTFIL15B (5' primer) and NCTFIL15F (3' primer) were used for the primaryamplification when maintenance of the mature sequence was desired.

    __________________________________________________________________________    Amino Acid                                                                    Clone                                                                              Substitutions                                                                          Expected                                                                              Primary PCR                                                                            Primary PCR                                    Name D56  Q156                                                                              Phenotype                                                                             5' Primer                                                                              3' Primer                                      __________________________________________________________________________    DQ   D    Q   mature  NTFIL15B NCTFIL15F                                        SQSQβ-/γ+D56SER5NCTFIL15F                                          DSDSβ+/γ-NTFIL15BQ156SER3                                          SSSSβ-/γ+D56SER5Q156SER3                                           CQCQβ-/γ+D56CYS5NCTFIL15F                                          DCDCβ+/γ-NTFIL15BQ156CYS3                                          CCCCβ-/γ+D56CYS5Q156CYS3                                         __________________________________________________________________________    Primer Name                                                                           Sequence                                                                 - Primary PCR                                                                D56Cys5(5'-AATGTAATAAGTTGTTTGAAAAAAATT-3')SEQ ID NO:3                         D56Ser5(5'-AATGTAATAAGTTCTTTGAAAAAAATT-3')SEQ ID NO:4                         Q156Cys3(5'-GTTGATGAACATGCAGACAATATG-3')SEQ ID NO:5                           Q156Ser3(5'-GTTGATGAACATAGAGACAATATG-3')SEQ ID NO:6                           NTFIL15B(5'-GTCCTCGCAACTAAGTCGACTAACTGGGT-SEQ ID NO:7                           GAATGTAATA-3')                                                              NCTFIL15F(5'-GAGTCATTCTCGACTTGCGGCCGCACCAG-SEQ ID NO:8                          AAGTGTTGATGAACAT-3'                                                          - Secondary PCR                                                              IL15PIXYF5(5'-AATATGGTACCTTTGGATAAAAGAGACTA-SEQ ID NO:9                         CAAGGACGACGATGACAAGAACTGGGTGAAT-                                              GTAATAAGT-3')                                                               IL15PIXY3(5'-GCGATATATCCATGGTCAAGAAGTGTTGA-SEQ ID NO:10                         TGAACAT-3')                                                           

Alternatively, oligonucleotide NTFIL15B could be substituted witholigonucleotide IL15PIXYF5, and oligonucleotide NCTFIL15F could besubstituted with oligonucleotide IL15PIXY3. Primary PCR amplificationwas performed in 100 μl of 1×Taq polymerase buffer (Boehringer)containing 250 μM dNTPs and 50 pmol of the 5' and 3' oligonucleotideprimer. The DNA template used was approximately 50 ng of pIXY764. VectorpIXY764 is similar to the above-described vector pIXY456 that containsDNA encoding human flag IL-15, wherein the N-linked glycosylation sitesof human IL-15 have been inactivated using procedures described supra.Reaction mixtures were overlaid with mineral oil, and heated to 94° C.in the thermal cycler for 5 minutes before the addition of 2 Units ofTaq polymerase (Boehringer) and the commencement of thermal cycling.Cycling conditions were denaturation at 94° C. for 45 seconds, annealingat 45° C. for 45 seconds and extension at 72° C. for 1 minute for atotal of 30 cycles.

Approximately 20 ng of gel purified product from the primaryamplification was used as the template for the secondary PCRamplification. All constructs were amplified with IL15PIXYF5 andIL15PIXY3 using the same buffer conditions as before. Cycling conditionswere denaturation at 94° C. for 45 seconds, annealing at 60° C. for 45seconds and extension at 72° C. for 1 minute, for a total of 20 cycles.

Amplification products were gel purified and digested with Asp718(Boehringer) and NcoI (New England Biolabs) overnight at 37° C. in1×Boehringer buffer B. The restriction products were ligated into apIXY456 yeast expression vector that had been digested with Asp718 andNcoI. This DNA was used to transform DH10β E. coli cells byelectroporation.

Plasmid DNA from single transfornants was sequenced to confirm sequenceintegrity, and used to transform XV2181 S. cerevisiae. Biologicalactivity was assayed using yeast supernatant following 30 hourinduction.

These experiments employed a PCR-based strategy for the mutagenesis onaccount of the mutagenesis sites being located near the ends of theIL-15 gene. However, these, and any other single or multiple pointmutations could be introduced by conventional site-directed mutagenesistechniques.

EXAMPLE 2 Monoclonal Antibodies Against IL-15

This example describes the method used to obtain three anti-IL-15monoclonal antibodies that function as antagonists of IL-15. All methodsused are conventional techniques, except where noted.

Balb/c mice were injected intraperitoneally on two occasions at 3 weekintervals with 10 μg of yeast-derived human IL-51 in the presence ofRIBI adjuvant (RIBI Corp., Hamilton. Mont.). Mouse sera was then assayedby conventional dot blot technique, antibody capture (ABC) andneutralization assay (CTLL-2 assay) to determine which animal was bestto fuse. Three weeks later, mice were given an intravenous boost of 3 μgof human IL-15 suspended in sterile PBS. Three days later, mice weresacrificed and spleen cells were fused with Ag8.653 myeloma cells (ATCC)following established protocols. Briefly, Ag8.653 cells were washedseveral times in serum-free media and fused to mouse spleen cells at aratio of three spleen cells to one myeloma cell. The fusing agent was50% PEG: 10% DMSO (Sigma). Fusion was plated out into twenty 96-wellflat bottom plates (Corning) containing HAT supplemented DMEM media andallowed to grow for eight days. Supernatants from resultant hybridomaswere collected and added to a 96-well plate for 60 minutes that had beenfirst coated with goat anti-mouse Ig. Following washes, ¹²⁵ I-IL-15 wasadded to each well, incubated for 60 minutes at room temperature, andwashed four times. Positive wells were subsequently detected byautoradiography at -70° C. using Kodak X-Omat S film. Positive cloneswere grown in bulk culture and supernatants were subsequently purifiedover a Protein A column (Pharmacia). The clones designated as M110, M111and M112 were each subsequently isotyped as IgG1 monoclonal antibodies.Hybridomas producing monoclonal antibodies M110, M111 and M112 have beendeposited with the American Type Culture Collection, Rockville, Md., USA(ATCC) on Mar. 13, 1996 and assigned accession numbers HB12061, HB-12062and HB-12063, respectively. All deposits were made according to theterms of the Budapest Treaty.

Monoclonal antibodies generated can be assayed for IL-15 antagonistactivity using the CTLL-2 assay as essentially described by Gillis, etal., Id.

EXAMPLE 3 Modified IL-15 Molecules

This example describes a method for obtaining modified IL-15moleculesthat function as IL-15 antagonists.

PEGylated IL-15

All conjugation reactions were performed with PEG, 5000 molecularweight, that was obtained in forms of succinimidyl succinate PEG(SS-PEG), succinimidyl carbonate PEG (SC-PEG), VS-PEG and Mal-PEG fromShearwater Polymers, Inc. (Huntsville, Ala.). Both of the SS-PEG andSC-PEG react with the .di-elect cons.-amino group of lysine, forming ahydrolytically unstable ester linkage in the case of SS-PEG, and ahydrolytically stable urethane linkage in the case of SC-PEG. PEGylationwas performed in 50 nM NaH₂ PO₄ at pH 9.0 for SS-PEG and SC-PEG; and atpH 7.0 for reactions containing VS-PEG and Mal-PEG. The reactionsproceeded in 0.5 ml volumes at 100 μg/ml. In each reaction, PEG wasadded to the reaction mixtures at molar ratios of PEG to lysine of 1:1,3:1, 10:1 and 100:1 (there are 9 lysine residues in each simian IL-15molecule). The reactions proceeded overnight at 4° C.

Characterization of PEGylated simian IL-15 was made by SDS-PAGE on 4-20%gradient polyacrylamide gels (Novex, San Diego, Calif.). Conventionalsilver staining techniques were used for unmodified IL-15 proteinsloaded at approximately 0.5 μg/lane. Highly PEGylated simian IL-15proteins required loading larger quantities of protein onto the gel forvisualization. Western blots were also used to characterize the highlyPEGylated IL-15. In these experiments, PEGylated simian IL-15 wasseparated by SDS-PAGE, transferred to nitrocellulose membrane, incubatedwith monoclonal antibody M111, followed by incubation with goatanti-mouse HRP, and finally visualized with 4 CN Membrane PeroxidaseSubstrate System (Kirkegaard & Perry Laboratories, Gaithersburg, Md.).PEGylated simian IL-15 was also characterized by size exclusionchromatography (SEC) HPLC with a Biosil SEC-250 sizing column (Biorad,Richmond, Calif.) according to conventional techniques.

SC-PEGylated FLAG-simian IL-15 was tested for its ability to bind totransfected COS cells that expressed IL-15 α-, or β- and γ-receptorsubunits on the cell membrane surface. The PEGylated IL-15 inhibitedradiolabeled IL-15 binding to COS cells expressing the IL-15R α-subunitindicating that PEGylated IL-15 competes for IL-15Rα-subunit binding.Further, the PEGylated IL-15 did not inhibit binding of radiolabeledIL-15 to COS cells expressing β- and γ-receptor subunits indicating thatthe PEGylated IL-15 does not bind to β- and/or γ-receptor subunits ofthe IL-15 receptor complex. Thus, PEGylated IL-15 prevents endogenousIL-15 from effecting signal transduction through the β- and γ-receptorsubunits of the IL-15 receptor complex.

EXAMPLE 4 Inhibition of IL-15 Activity in CTLL-2 Assay

This example further illustrates a method for determining the preventionby the antagonists according to the invention of signal transduction ofIL-15 through the β- and γ-receptor subunits of the IL-15 receptorcomplex.

Antagonist activity of monoclonal antibodies, PEGylated IL-15 and IL-15muteins can be assessed using a modified CTLL-2 cell ³ H-Thymidineincorporation assay (Gillis, et al., Id.). Serial dilutions ofantagonist can be made in 96-well flat-bottom tissue culture plates(Costar, Cambridge, Mass.) in DMEM medium (supplemented with 5% FCS,NEAA, NaPyruvate, HEPES pH 7.4, 2-me, PSG) at a final volume of 50 μl. Asub-optimal amount of IL-15 (final concentration of 20-40 pg/ml) then isadded to all assay wells (5 μl/well) after serial dilution of samplesand prior to addition of cells. Washed CTLL-2 cells are added (about2000 per well in 50 μl) and the plates are incubated for 24 hours at 37°C. in a humidified atmosphere of 10% CO₂ in air. This was followed by afive hour incubation with 0.5 μCi of ³ H-Thymidine (25 Ci/Mol. Amersham,Arlington Heights, Ill.). The cultures then are harvested on glass fiberfilters and counted by avalanche gas ionization either on amultidetector direct beta counter (Matrix 96, Packard InstrumentCompany, Meridien, Conn.) or on a beta scintillation counter. The countsper minute (CPM) generated by the assay are converted to percentinhibition and the percent inhibition values of each titrated antagonistsample are used to calculate antagonist activity in units/ml.

Data showing the concentration needed to neutralize 40 pg/ml of IL-15 ina CTLL inhibition assay is provided in Table I below. Table II belowshows the activity of IL-15 (agonist activity) and IL-15 antagonists inCTLL and CTLL inhibition assays.

                  TABLE I                                                         ______________________________________                                        Specific Activity of IL-15 Antagonists                                          The concentration of antagonist required to neutralize 40 pg/ml IL-15       in                                                                              CTLL inhibition assay:                                                        antagonist   concentration                                                                             method of protein determination                    ______________________________________                                        huIL-15 muteins                                                                          848-2560 pg/ml                                                                            ELISA/estimated from AAA                                 M110, M111 5 ng/ml OD                                                         PDGhuIL-15 D56C 7.7 ng/ml estimated from AAA                                  M112 40 ng/ml OD                                                              PDGf-s-IL15 140-196 ng/ml AAA                                               ______________________________________                                         OD = optical density absorbence at 280 nm; extinction coefficient of 1.35     AAA = amino acid analysis                                                     PEGfs-IL15 = PEGylated flag simian IL15                                  

                  TABLE II                                                        ______________________________________                                        Activity of IL-15 and IL-15 Antagonists                                         in CTLL and CTLL Inhibition Assays                                                          CTLL Assay   CTLL Inhibition Assay                               units/ml units/ml                                                            sample (Agonist Activity) (Antagonist Activity)                             ______________________________________                                        IL-15       7.09 × 10.sup.5                                                                      279                                                    IL-15-Q156C -- 3 × 10.sup.6                                             IL-15-Q156S -- 1.5 × 10.sup.6                                           IL-15-D56C -- 2 × 10.sup.6                                              IL-15-D56C-Q156C -- 2 × 10.sup.5                                        IL-15-D56C-Q156S -- 7 × 10.sup.5                                        IL-15-D56S -- 2.2 × 10.sup.5                                            IL-15-D56S-Q156S -- 7.2 × 10.sup.5                                      vector control -- 1141                                                        IL-15 3.7 × 10.sup.8 NA                                                 PEG-IL-15 -- 2.3 × 10.sup.6                                             PEG-IL-15-D56C -- 7.96 × 10.sup.6                                       IL-15-D56C -- 5 × 10.sup.6                                              IL-15 5.6 × 10.sup.8 NA                                                 PEG-IL-15 NA 1.7 × 10.sup.5                                           ______________________________________                                         Q156C = Gln.sup.156 substituted with Cys                                      Q156S = Gln.sup.156 substituted with Ser                                      D56C = Asp.sup.56 substituted with Cys                                        D56S = Asp.sup.56 substituted with Ser                                        NA: not assayed                                                          

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..342                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG AGA ATT TCG AAA CCA CAT TTG AGA AGT AT - #T TCC ATC CAG TGC        TAC       48                                                                    Met Arg Ile Ser Lys Pro His Leu Arg Ser Il - #e Ser Ile Gln Cys Tyr            1               5 - #                 10 - #                 15              - - CTG TGT TTA CTT CTA AAG AGT CAT TTT CTA AC - #T GAA GCT GGC ATT CAT           96                                                                       Leu Cys Leu Leu Leu Lys Ser His Phe Leu Th - #r Glu Ala Gly Ile His                        20     - #             25     - #             30                  - - GTC TTC ATT TTG GGC TGT TTC AGT GCA GGG CT - #C CCT AAA ACA GAA GCC          144                                                                       Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Le - #u Pro Lys Thr Glu Ala                    35         - #         40         - #         45                      - - AAC TGG GTG AAT GTA ATA AGT GAT TTG AAA AA - #A ATT GAA GAT CTT ATT          192                                                                       Asn Trp Val Asn Val Ile Ser Asp Leu Lys Ly - #s Ile Glu Asp Leu Ile                50             - #     55             - #     60                          - - CAA TCT ATG CAT ATT GAT GCT ACT TTA TAT AC - #A GAA AGT GAT GTT CAC          240                                                                       Gln Ser Met His Ile Asp Ala Thr Leu Tyr Th - #r Glu Ser Asp Val His            65                 - # 70                 - # 75                 - # 80       - - CCC AGT TGC AAG GTA ACA GCA ATG AAG TGC TT - #T CTC TTG GAG TTG CAA          288                                                                       Pro Ser Cys Lys Val Thr Ala Met Lys Cys Ph - #e Leu Leu Glu Leu Gln                            85 - #                 90 - #                 95              - - GTT ATT TCA CAT GAG TCC GGA GAT ACA GAT AT - #T CAT GAT ACA GTA GAA          336                                                                       Val Ile Ser His Glu Ser Gly Asp Thr Asp Il - #e His Asp Thr Val Glu                       100      - #           105      - #           110                  - - AAT CTT ATC ATC CTA GCA AAC AAC ATC TTG TC - #T TCT AAT GGG AAT ATA          384                                                                       Asn Leu Ile Ile Leu Ala Asn Asn Ile Leu Se - #r Ser Asn Gly Asn Ile                   115          - #       120          - #       125                      - - ACA GAA TCT GGA TGC AAA GAA TGT GAG GAA CT - #A GAG GAA AAA AAT ATT          432                                                                       Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Le - #u Glu Glu Lys Asn Ile               130              - #   135              - #   140                          - - AAA GAA TTT TTG CAG AGT TTT GTA CAT ATT GT - #C CAA ATG TTC ATC AAC          480                                                                       Lys Glu Phe Leu Gln Ser Phe Val His Ile Va - #l Gln Met Phe Ile Asn           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - ACT TCT TGA              - #                  - #                      - #        489                                                                 Thr Ser                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 489 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..489                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - ATG AGA ATT TCG AAA CCA CAT TTG AGA AGT AT - #T TCC ATC CAG TGC TAC           48                                                                       Met Arg Ile Ser Lys Pro His Leu Arg Ser Il - #e Ser Ile Gln Cys Tyr             1               5 - #                 10 - #                 15              - - TTG TGT TTA CTT CTA AAC AGT CAT TTT CTA AC - #T GAA GCT GGC ATT CAT           96                                                                       Leu Cys Leu Leu Leu Asn Ser His Phe Leu Th - #r Glu Ala Gly Ile His                        20     - #             25     - #             30                  - - GTC TTC ATT TTG GGC TGT TTC AGT GCA GGG CT - #T CCT AAA ACA GAA GCC          144                                                                       Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Le - #u Pro Lys Thr Glu Ala                    35         - #         40         - #         45                      - - AAC TGG GTG AAT GTA ATA AGT GAT TTG AAA AA - #A ATT GAA GAT CTT ATT          192                                                                       Asn Trp Val Asn Val Ile Ser Asp Leu Lys Ly - #s Ile Glu Asp Leu Ile                50             - #     55             - #     60                          - - CAA TCT ATG CAT ATT GAT GCT ACT TTA TAT AC - #G GAA AGT GAT GTT CAC          240                                                                       Gln Ser Met His Ile Asp Ala Thr Leu Tyr Th - #r Glu Ser Asp Val His            65                 - # 70                 - # 75                 - # 80       - - CCC AGT TGC AAA GTA ACA GCA ATG AAG TGC TT - #T CTC TTG GAG TTA CAA          288                                                                       Pro Ser Cys Lys Val Thr Ala Met Lys Cys Ph - #e Leu Leu Glu Leu Gln                            85 - #                 90 - #                 95              - - GTT ATT TCA CTT GAG TCC GGA GAT GCA AGT AT - #T CAT GAT ACA GTA GAA          336                                                                       Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Il - #e His Asp Thr Val Glu                       100      - #           105      - #           110                  - - AAT CTG ATC ATC CTA GCA AAC AAC AGT TTG TC - #T TCT AAT GGG AAT GTA          384                                                                       Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Se - #r Ser Asn Gly Asn Val                   115          - #       120          - #       125                      - - ACA GAA TCT GGA TGC AAA GAA TGT GAG GAA CT - #G GAG GAA AAA AAT ATT          432                                                                       Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Le - #u Glu Glu Lys Asn Ile               130              - #   135              - #   140                          - - AAA GAA TTT TTG CAG AGT TTT GTA CAT ATT GT - #C CAA ATG TTC ATC AAC          480                                                                       Lys Glu Phe Leu Gln Ser Phe Val His Ile Va - #l Gln Met Phe Ile Asn           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - ACT TCT TGA              - #                  - #                      - #        489                                                                 Thr Ser                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - AATGTAATAA GTTGTTTGAA AAAAATT          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - AATGTAATAA GTTCTTTGAA AAAAATT          - #                  - #                 27                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GTTGATGAAC ATGCAGACAA TATG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GTTGATGAAC ATAGAGACAA TATG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - GTCCTCGCAA CTAAGTCGAC TAACTGGGTG AATGTAATA      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GAGTCATTCT CGACTTGCGG CCGCACCAGA AGTGTTGATG AACAT   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AATATGGTAC CTTTGGATAA AAGAGACTAC AAGGACGACG ATGACAAGAA  - #                  50                                                                         - - CTGGGTGAAT GTAATAAGT             - #                  - #                    69                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GCGATATATC CATGGTCAAG AAGTGTTGAT GAACAT      - #                  -     #       36                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid that encodes a mutein ofIL-15 capable of binding to the IL-15R α-subunit and incapable oftransducing a signal through the β- or γ-subunits of the IL-15 receptorcomplex, wherein the mutein comprises at least one addition,substitution, or deletion that interferes with binding of (a) amino acidAsp⁵⁶ of the IL-15 molecule to the β-subunit of the IL-15 receptorcomplex or (b) amino acid Gln156 of the IL-15 molecule to the γ-subunitof the IL-15 receptor complex.
 2. An isolated nucleic acid according toclaim 1, wherein the mutein of IL-15 has at least one of the amino acidresidues Asp⁵⁶ or Gln¹⁵⁶ deleted or substituted with a differentnaturally-occurring amino acid residue.
 3. An isolated nucleic acidaccording to claim 2, wherein either or both of Asp⁵⁶ and Gln¹⁵⁶ areeach substituted with a serine or cysteine.
 4. An isolated nucleic acidaccording to claim 2, wherein Asp⁵⁶ is substituted with serine orcysteine.
 5. An isolated nucleic acid according to claim 2, whereinGln¹⁵⁶ is substituted with serine or cysteine.
 6. A recombinant vectorthat comprises a nucleic acid of claim
 1. 7. A host cell transformed ortransfected with the vector of claim
 6. 8. A method of producing anIL-15 mutein comprising culturing a host cell according to claim 7 underculture conditions that are conducive to expression of such IL-15mutein.